In recent years, studies using proteomic approaches have been conducted actively as post-genomic research. This is because proteins as gene products are probably related more directly to the state of disease than genes. Therefore, proteomic analysis is expected to be capable of discovering many pathogenic proteins or disease-related factors undiscoverable by genomic analysis. For example, the proteomic analysis facilitates discovery of biomarker proteins induced or deleted by particular disease. The biomarkers behave in relation to the disease state and as such, highly possibly serve as diagnostic markers or targets for drug development/discovery. Furthermore, the biomarkers lead to the direct interests of patients, such as evaluation of drug responsiveness or prediction of adverse reaction onset.
Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometers (MALDI-TOF-MS), MS/MS mass spectrometers (tandem mass spectrometers), liquid chromatography mass spectrometers (LC-MS mass spectrometers), and the like have been put to practical use by virtue of the improved performance of mass spectrometers (MS). With such advances of technology, the proteomic analysis has achieved high-speed structural analysis of proteins, etc. as well as high-throughput ultramicroanalysis of polypeptides or identification of previously undetectable, very low-abundance proteins, and has become a strong tool for search for disease-related factors.
However, the proteomic analysis of body fluid samples, particularly serum and plasma, is behind that targeted for biological tissues, in spite of its big clinical advantage. This is because, for example, the abundance of major proteins such as albumin or globulin exceeds approximately 99% of all serum or plasma proteins (Non-Patent document 1), and along with removal of these proteins, the majority of low-molecular-weight proteins/peptides components are also lost.
Pretreatment techniques of removing major proteins in serum or plasma have been developed so far, including: a method comprising obtaining a solution by removing excessive proteins that hamper the detection of low-abundance components (Patent Documents 1 and 2); a method comprising concentrating a fractionated protein solution using a plurality of electrodes (Patent Document 3); and a method for removing major proteins in serum, comprising precipitating large proteins using an organic solvent, and dissociating therefrom low-molecular-weight proteins (Non-Patent Document 2).
However, along with removal of the major proteins, low-molecular-weight proteins/peptides interacting therewith are also lost. Therefore, it has still been demanded to develop a method for concentrating low-molecular-weight proteins/peptides in a body fluid sample, particularly, in serum or plasma, with high efficiency and good reproducibility without being influenced by major proteins.    Patent Document 1: Japanese Patent Laid-Open No. 2005-126376    Patent Document 2: Japanese Patent Laid-Open No. 2005-156249    Patent Document 3: Japanese Patent Laid-Open No. 2007-139759    Non-Patent Document 1: Tirumalai et al., Mol. Cell. Proteomics 2.10, 1096-1103, 2003    Non-Patent Document 2: Merrell et al., Journal of Biomolecular Techniques 15: 238-248, 2004